Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos.
BACKGROUND
The dual-luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism.


RESULTS
Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the in vivo response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase) and inducible (NF-kappaB-dependent) promoters. The flexibility of this assay is demonstrated by induction of the NF-kappaB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown.


CONCLUSIONS
This assay has several advantages compared with the classical in vitro (cell lines) and in vivo (transgenic mice) approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.
Alcaraz-Pérez
Francisca
F
Research Unit, Department of Surgery, University Hospital Virgen de la Arrixaca, 30120 Murcia, Spain. palcaraz@um.es
Mulero
Victoriano
V
Cayuela
María L
ML
D000818:Animals; D047108:Embryonic Development; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D049409:Luciferases, Firefly / Q000235:genetics* / Q000378:metabolism; D049410:Luciferases, Renilla / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020798:Two-Hybrid System Techniques; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism
D016328:NF-kappa B; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly